1. Genomics Collaboration Senior Scientist
Genome-Wide Homozygosity Mapping and Linkage Analysis in Consanguineous Families Using the Affymetrix Mapping 10K Array Identifies a Novel Deafness Locus
Carsten Rosenow, Ph.D.
Genomics Collaboration Senior Scientist
Genotyping

2. Systems Biology Workflow
Mapping
10K Array
Expression
Array
Identify
candidate
regions
Linkage
Fine Mapping
Differentially
Expressed Genes
Clinical analysis
Functional analysis
CustomSeq

3. Genetic Analysis Workflow
Analysis Tool
Experimental Approach
Mapping10K
Linkage
LOH
HBD
Fine Mapping
Two Affymetrix products can be used for Genetic analysis. First the Mapping 10K array for applications like Linkage, LOH and HBD (Homozygosity by descent) and second the CustomSeq array for sequence analysis and novel SNP discovery. Candidate regions identified by either Expression analysis or Mapping 10K analysis can be used mapped using the CustomeSeq platform
Sequence Analysis
Novel SNP discovery
CustomSeq™

4. GeneChip Mapping 10K

5. Presentation overview
Technical Overview
Application:
Homozygosity by descent analysis
Linkage Mapping
Data Analysis
Spotfire decision site

6. PCR: One Primer Amplification
GeneChip® Mapping 10K Assay Overview
250 ng Genomic DNA
Xba
RE Digestion
Adaptor
Ligation
PCR: One Primer Amplification
Complexity
Reduction
The new developed assay for the mapping 10K array is the cornerstone of the product. Traditionally genotyping was conducted using locus specific PCR. Since this approach is not scalable we have developed a novel assay tgo map more than SNPs using a complexity reduction and amplification schema.
The Mapping assay begins with only 250 ng genomic DNA.
It is digested with a restriction enzyme, such as XbaI and we are going to get a whole range of sizes
We ligate a common set of adaptors, which will ligate to all the size fragments.
Next, a PCR step is optimized to only amplify fragments in the bp range (as shown in red in the first fragment).
Followed by fragmentation, labeling, and hybridization on the same platform used for expression analysis.
We have selected our SNPs from the TSC database based on their presence in this “Xba” fraction of the genome. (see next few)
Fragmentation
and Labeling
AA BB AB
Hyb & Wash

7. GeneChip Mapping 10K Tiling Strategy
High probe redundancy
40 probes (PM and MM) per SNP
20 probes per SNP allele
10 probes per DNA strand
Forward and reverse strand interrogated
An important feature of our accuracy is probe redundancy. Historically we interrogate every gene with currently 11 probes which gives us a good estimate of gene expression. Accuracy in Genotyping is an important requirement since genotype errors can lead to inflated or deflated lod scores. We use 40 probes for each SNP interrogated. Each position has a perfect match and mismatch probe. 20 probes per allele or 10 per strand. We interrogate forward and reverse strand.

8. GeneChip Mapping 10K tiling strategy
Allele 1 C G
Forward
Reverse
SNP position T A -3 -1 +2 +4
Allele 1 C G
Forward
Reverse
Allele 2 A T
In addition to the center or SNP position we interrogate neighboring bases (from -4 to +4) with probes consisting of perfect match and mismatch probes.

9. Genome Coverage: 400 Microsatellite Markers
Dec 2002 Golden Path
Dec 2002 Golden Path
Blue = STR from CIDR panel
Black = Gaps
Red = at least 1 SNP per 100 kb
Black = Gaps
Median intermarker distance: Mb
Mean intermarker distance: Mb
Mean genetic gap distance: cM
Average Heterozygosity
Here is the coverage of the 400 marker set used by CIDR.
Note every chromosome has markers, but it is somewhat uneven.
Black represent GAPs in the human genome, where there is no sequence.
Median intermarker distance: kb
Mean intermarker distance: kb
Mean genetic gap distance: cM
Average Heterozygosity

10. Linkage analysis Information content:
Measurement of the goodness of a set of markers
Function of marker heterozygosity and number of meiosis in a study
For multipoint linkage analysis marker density is also important
Nuclear families

11. Information content comparison Affymetrix Mapping 10K - Marshfield

12. SNP Annotation NetAffx™ Analysis Center

13. Homozygosity by Descent study (HBD)
Molecular Otolaryngology Research Laboratories, University of Iowa
Richard JH Smith
Nicole Meyer

14. Homozygosity by descent (HBD) mapping for rare autosomal recessive disorders
Basic principle of homozygosity by descent. Each parent gives one chromosome to an offspring. In an inbreeding family the ancestor allele (here in yellow) gets inherited by the affected child since both parents are carrier of the allele. If this allele contains a recessive SNP the child would be affected (recessive homozygote). In addition to linkage analysis the children can be analysed for large regions of homozygosity by descent for the identification of the disease causing allele.
HBD

15. Homozygosity mapping for rare disorders
In rare autosomal recessive diseases, inbred pedigrees are highly informative
When parents are consanguineous, affected children are homozygous for alleles identical by descent (IBD)
One affected child of a 1st cousin marriage is as informative as 3 affected children of unrelated parents
Linkage mapping can be carried out by searching for regions of increased marker homozygosity
Single pedigree can be powerful enough to map rare recessive traits

16. Study Consanguineous family studied
Four out of five children are affected with deafness
Hereditary deafness affects 1:2,000 newborns and accounts for greater than 50% of severe-to-profound childhood deafness

17. Workflow Type >10K genotypes in the family
parents, affected and unaffected children
Mendel inheritance error check
replace Mendel error with nocall
Homozygosity by descent (HBD)
high stringency analysis
treat no calls as homozygous regions
look for homozygous regions in all affected individuals
Disregard regions which are also homozygous in parents and in the unaffected children
Identify ancestor haplotypes

18. Pedigree
Autosomal recessive disease

19. Analysis Analysis done using 400 microsattelite markers
HBD region identified on Chromosome 1
Analysis done with GeneChip Mapping 10K array
HBD region on Chromosome 1 confirmed
Novel HBD locus identified on Chromosome 3
Lod scores for both regions is 2.8

20. Chromosome 1: HBD region confirmed
PARENTS 3 4 5 6 7 1
SNP1 2
MS1
SNP2
MS2
MS3
MS4
MS5
SNP3
SNP4
SNP5
SNP6
MS6
MS7
SNP7
SNP8
SNP9
SNP10
SNP11
SNP12
SNP13
SNP14

21. Chromosome 3
Homozygosity by descent

22. Homozygosity by Descent Analysis in Spotfire Decision Site

23. Import raw data
Import raw data from database, text files, or clipboard
Easily join patient phenotype information

24. Utilize annotations from NetAffx http://www. affymetrix
NetAffx provides annotation files with known SNP attributes (e.g. chromosome, location, etc…)

25. Annotations enable visualization
Each blue line represents one SNP

26. Perform HBD calculations (1)
To start, count instances of homozygosity among affected individuals.
Marked records below show two SNPs where 4 of 4 individuals are homozygous for this allele.

27. Perform HBD calculations (2)
Generate statistical significance of homozygosity over “sliding” window of user-defined size along chromosomes.

28. Visualize results (1)
Visualize chromosome map using color and size to reflect regions of significant homozygosity.

29. Visualize results (2)
Or use slider to focus on most relevant SNPs, mark, and add column as reminder.

30. Visualize results (3)
Visualize the significance over the chromosome using the line chart, and drill down to specific locations.

31. Incorporate known genetic markers
Add known disease markers from OMIM and visualize alongside SNPs (genes as dark blue marks)

32. Are relevant genes near significant SNPs?
Step 1: Focus on area of significance on chromosome 3 (zoom using range sliders)
(zoom range)

33. Are relevant genes near significant SNPs?
Step 2: Filter down to relevant genes (blue x’s) using text search query device
Filter
Closest known deafness locus
~15 x 106 bp
Conclusion: New deafness loci is yet to be discovered in this region

34. Incorporate metrics from other software
By manually parsing and importing the results from MERLIN linkage software, one can visualize linkage results. (same significant region on chr 3)

35. Acknowledgements
Molecular Otolaryngology Research Laboratories, University of Iowa
Richard JH Smith
Nicole Meyer
Affymetrix
Shoulian Dong
Rui Mei
Spotfire
Brendan Gibson
Kristen Stoops